Influenza A virus is a member of the orthomyxoviridae virus family of (−)-sense RNA viruses. The influenza A viral genome is composed of 8 segments or chromosomes which encode 11 proteins1. During infection, the virus-encoded RNA-dependent RNA polymerase (RdRP) converts the (−)-stranded RNAs to (+) strand messenger RNAs and a set of full length complementary genomic RNAs (or cRNAs) which serve as templates for genomic replication. Viral proteins expressed from the (+) strand messenger RNAs go about the task of establishing infection and facilitating viral replication, a process which ends in the amplification, assembly, and release of virus particles containing the initial 8 (−) strand chromosomes which repeat the infectious cycle.
The processes associated with the transcription and replication of the influenza A genome have been under investigation for decades. All eight chromosomes of every influenza A strain (including H1N1 seasonal, H1N1 “swine”, H3N2, and H5N1 “avian”) contain identical 5′ and nearly identical 3′ untranslated regions (UTRs) flanking the protein-coding portion of the sequence which otherwise encode distinct proteins. Experimental results demonstrate that the UTRs are recognized by RdRP as a promoter element and both components are critical for viral gene expression and replication. Hence, the viral polymerase and its cognate UTR RNA ligand are thought to control the viral life cycle and are critical targets for therapeutic intervention.
Despite such relatively detailed knowledge of viral replication, currently existing and clinically approved anti-influenza A therapeutic molecules are restricted to small molecules that inhibit one of two target classes of viral coat proteins: Neuraminidase (NA) and Matrix 2 (M2). NA belongs to a broad class of glycoside hydrolase enzymes (also known as sialidases, as N- or O-linked neuraminic acids are collectively called sialic acid) which cleave terminal sialic acid residues off virions and host cell receptor proteins. During influenza A infection, NA activity is involved in viral transit through mucus secretions of the respiratory tract as well as for the biochemical separation/elution of secreted viruses from the infected cells serving as sites of replication, thereby enabling the infection of nearby healthy cells. Currently, oseltamivir (trade name Tamiflu®) and zanamivir (trade name Relenza®) are two clinically approved medications for the treatment of influenza infection through inhibition of NA while laninamivir (Inavir) and peramivir are currently in the late stages of clinical trials as next-generation influenza NA inhibitors.
Unlike NA, influenza M2 is produced by the alternative splicing of the mRNA encoding the viral structural protein, matrix (or M). In contrast to M which is one of the most highly abundant viral proteins in infection and serves as a scaffold to which viral coat proteins and ribonucleoprotein particles bind, M2 is present in minute amounts in virions and serves as an ion channel which enables viral uncoating and escape from endosomal compartments into the cellular cytoplasm, a critical step in replication. Clinically approved inhibitors of M2 include amantadine (trade name Symmetrel®) and rimantadine (trade name Flumadine®).
NA and M2 inhibitors serve as successful proof-of-concept small molecule inhibitors of critical steps in the influenza virus infection cycle. Despite their early successes, however, use of both NA and M2 inhibitors has met recent challenges in treating influenza infection through the emergence of viral variants exhibiting drug resistance. For example, evidence of viral resistance to Tamiflu has been documented in numerous clinically relevant influenza A isolates including the 2009 pandemic5, H3N26, H5N17, and seasonal H1N18 where resistance existed in 99.6% of circulating isolates in 2008. Although influenza A resistance to zanamivir has yet to be reported, the report of an influenza B viral isolate resistant to this agent9 may indicate the possibility of future more prevalent resistance to this drug upon broad usage to treat influenza infections although the resistant virus described in this study had acquired an additional compensatory mutation in the HA protein which should reduce this possibility significantly. With regards to the M2 inhibitors, the Centers for Disease Control and Prevention indicated that nearly all circulating H3N2 and pandemic H1N1 virus isolates during the fall of 2009 were resistant to amantadines10.
Unfortunately, surveillance efforts have also identified the recent emergence of dual oseltamivir/adamantane resistant isolates11 indicating the need for the discovery and utilization of additional small molecule inhibitors targeting other critical viral targets. Thus, there is still a need to provide new and/or improved antiviral drugs that interfere with viral replication, and especially RNA-virus replication.